GS-2: A Novel Broad-Spectrum Agent for Environmental Microbial Control

The environmental control of microbial pathogens currently relies on compounds that do not exert long-lasting activity on surfaces, are impaired by soil, and contribute to the growing problem of antimicrobial resistance. This study presents the scientific development and characterization of GS-2, a novel, water-soluble ammonium carboxylate salt of capric acid and L-arginine that demonstrates activity against a range of bacteria (particularly Gram-negative bacteria), fungi, and viruses. In real-world surface testing, GS-2 was more effective than a benzalkonium chloride disinfectant at reducing the bacterial load on common touch-point surfaces in a high-traffic building (average 1.6 vs. 32.6 CFUs recovered from surfaces 90 min after application, respectively). Toxicology testing in rats confirmed GS-2 ingredients were rapidly cleared and posed no toxicities to humans or animals. To enhance the time-kill against Gram-positive bacteria, GS-2 was compounded at a specific ratio with a naturally occurring monoterpenoid, thymol, to produce a water-based antimicrobial solution. This GS-2 with thymol formulation could generate a bactericidal effect after five minutes of exposure and a viricidal effect after 10 min of exposure. Further testing of the GS-2 and thymol combination on glass slides demonstrated that the compound retained bactericidal activity for up to 60 days. Based on these results, GS-2 and GS-2 with thymol represent a novel antimicrobial solution that may have significant utility in the long-term reduction of environmental microbial pathogens in a variety of settings.

Introduction
To date, hospital-acquired infections account for an estimated 1.7 million infections and 99,000 deaths annually [1]. While many pathogens originate from patients, an estimated 20-40% of nosocomial infections are caused by cross-contamination from the hands of healthcare workers [2]. This cross-contamination likely results from healthcare personnel touching contaminated surfaces that are sanitized with agents that lack adequate antimicrobial activity [3]. Indeed, the current SARS-CoV-2 pandemic has drawn renewed attention to the environmental control of microorganisms and the role contaminated surfaces play in the spread of infectious diseases. One study demonstrated that SARS-CoV-2 can survive at room temperature on hard surfaces for at least 28 days [4] and on hands for approximately GS-2 activity was tested against four clinically isolated pathogens: MRSA, Streptococcus pyogenes, and the carbapenem-resistant enterobacteriaceae (CRE) E. coli and K. pneumoniae (samples generously donated from Kalispell Regional Medical Center, Montana) using a 96-well microplate format under CLSI M26-A guidelines [32]. A serial dilution in Oxoid Sensitest medium of GS-2 was combined with bacteria to determine the MIC and MBC of GS-2 after 24 h of incubation. MIC was determined by visual assessment with no turbidity. MBC was determined by combining technical replicates and centrifuging to pellet bacteria. The supernatant was removed, and the pellet was rehydrated with 1 mL sterile saline. One hundred microliters of this solution was plated onto 5% sheep blood agar (SBA) plates and incubated for 24 h at 37 • C. MBC was the concentration of GS-2 that produced a 99.99% reduction from the average starting inoculum. This value was calculated by a manual dilution series of the inoculum on 5% SBA plates (the starting inoculum was diluted, plated, incubated for 24 h, and counted to obtain an accurate starting concentration for the percent reduction calculation). For growth and contamination controls, media-only and water controls were set up on the 96-well plate and processed consistently with experimental samples to ensure sterility and quality control. Positive growth control wells containing bacteria, media, and sterile water were diluted, plated, and counted to determine the amount of growth from the starting inoculum and to ensure robust growth against the test organism.

Testing GS-2 and GS-2 with Thymol under Regulatory Disinfectant Standards
MRSA and E. coli (clinical isolates) were tested against GS-2 and GS-2 with thymol according to the Therapeutic Goods Administration (TGA) Option C household/commercialgrade disinfectant guidelines [33]. Briefly, bacteria were grown to log-phase in Oxoid Sensitest broth at 37 • C under constant rotation for 6 h. Three milliliters of either 15 mg/mL GS-2, DMSO, 5 mg/mL thymol in DMSO, 30 mg/mL GS-2 in DMSO, or 30 mg/mL GS-2 with 5 mg/mL thymol was combined with 9 × 10 8 CFU/mL bacteria in 1 mL and incubated for 8 min at room temperature. After 8 min, samples were vortexed, and 20 µL was placed in 5 mL Oxoid Sensitest medium and incubated for 48 h. A pass result required 2 or more of the 5 tubes to be clear (indicating no growth) after 48 h.

Antibacterial Time-kill Studies with GS-2 against Potential Bioterrorism Pathogen Surrogates
Cultures of Burkholderia thailandensis (ATCC 700388), a surrogate for Burkholderia pseudomallei, and Bacillus anthracis Sterne 34F2 (Fort Dodge Australia Pty Ltd., Colorado Serum Company, Denver, CO, USA) were prepared in nutrient broth and incubated overnight at 37 • C with shaking. Cultures were diluted to an OD 600 equivalent of 2 × 10 6 CFU/mL in fresh broth and incubated further for 1 h with shaking to obtain a log-phase culture. A final bacterial inoculum of 1 × 10 6 CFU/mL in 0.5 mL of the log-phase culture was added to 0.5 mL of broth containing two-fold-concentrated GS-2 (final concentration 4.76 mg/mL), ciprofloxacin (final concentration 0.01 mg/mL), or meropenem (final concentration 0.10 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). Cultures were incubated for 1 h, 3 h, 5 h, and 24 h (B. thailandensis) or 2 h, 4 h, 6 h, and 24 h (B. anthracis). Following each time-point, samples were washed in PBS, serially diluted, and plated onto nutrient agar plates. The plates were incubated at 37 • C for 24-48 h, and CFU/mL was calculated to determine time-kill kinetics.

Determining Antiviral Activity of GS-2 and GS-2 with Thymol
GS-2 was tested against Murine Hepatitis Virus Strain 1 (MHV-1; ATCC/VR-261, the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) surrogate), Herpes Simplex Virus-1 (HSV-1; ATCC/VR-733), and poliovirus (ATCC/VR-192) according to TMCV 006 ASTM International Standard E1053 [34]. Briefly, viral suspensions were prepared in 5% FBS, spread over the surface of a glass petri dish, and allowed to dry. Two milliliters of GS-2 (15 mg/mL) or GS-2 (30 mg/mL) with thymol (5 mg/mL) was then applied to the viral film and incubated for 10 min at RT. Following incubation, viruses were harvested from test and control slides with a cell scraper, and GS-2 or GS-2 with thymol was neutralized with 2% FBS in minimum essential medium (MEM). Separately, 2 mL GS-2 or GS-2 with thymol was also combined with 2% FBS in MM and then either combined with viral suspension to ensure neutralization (neutralization control) or set aside for the following step (cytotoxicity control).
The neutralized harvested test suspensions, neutralization control, and cytotoxicity control were serially diluted and plated in quadruplicate onto a 96-well plate containing a monolayer of either A9 cells (ATCC/CCL-1.4; for MHV-1) or Vero cells (ATCC/CCL-81; for HSV-1 and poliovirus). Four wells containing cells only served as controls. Media was added to all wells and incubated for 7-10 days. Then, each well was examined for the presence of cytopathic effects of infection, such as cell rounding, sloughing, and monolayer degradation. Cytotoxicity control wells were examined for damage caused by the test product. The Reed and Muench LD 50 method was used to determine the viral titer endpoint.

GS-2 Surface Sanitizer Trial
Two commercial high-rise buildings with near-identical layouts (built in the same year within 0.25 km of each other and monitored for daily traffic in the building with no significant differences in work population over the two-week test period) were sanitized for two weeks. Building A was sanitized with GS-2 at 15 mg/mL (1.5%), and building B was sanitized with a hospital-grade disinfectant containing 0.42% BAC according to product label instructions. After two weeks of treatment, swabs were taken in both buildings of five high-touch-point surfaces: loading dock bathroom door handles, office bathroom door handles for one level, office door handles into the loading dock, elevator buttons, and elevator touch screens (identical in both buildings). Swabs were taken 90 min before the morning treatment, 90 min after the morning treatment, and again at 4 h after treatment. Swabs were then cultured on 5% sheep blood agar (SBA) plates (Thermo Fisher Scientific, Scoresby, Australia) for 24 h at 37 • C, and the number of colonies was counted.

Toxicology Studies with GS-2
All animal handling and treatment was approved by the University of Melbourne Animal Ethics Committee. All animals were given free access to food, water, and enrichment. They were co-housed for a 72 h acclimation period prior to the beginning of experiments and then housed singly during the dosing protocol. Eight Sprague-Dawley rats were given a subcutaneous bolus dose of GS-2 at 10 mL/kg under the skin between the shoulder blades. Tail vein blood was drawn, processed, and assayed for capric acid at 1 h, 4 h, 8 h, and 24 h via LC-MS. During this period, the rats were observed for any adverse events or abnormal behavior. At 24 h, the animals were euthanized.

Testing Residual Surface Activity of GS-2 with Thymol
Sterile glass slides were coated with either 200 µL GS-2 with thymol or 200 µL sterile water and allowed to dry. On days 0, 15, 30, and 60, slides were inoculated with 1 × 10 6 CFUs MRSA or P. aeruginosa and incubated for 60 min, uncovered, at room temperature. Viable bacteria were then extracted from the slides, diluted, plated onto 5% SBA plates, incubated overnight, and colonies were counted. Enumeration was performed as per NCCLS/CLSI guideline M26-A.

Statistics
Statistical analyses (Student's unpaired two-tailed t-test) were performed with Graph-Pad Prism 9.0 software (GraphPad Software, San Diego, CA, USA).

GS-2 Development and Characterization
Capric acid is a 10-carbon saturated fatty acid with known antibacterial and antifungal properties [36][37][38]. However, esterifying fatty acids to solubilize them has been shown to reduce activity [24,30]. Our initial goal was to discover a method for solubilizing the fatty acid without compromising the native structure. We also needed to solubilize the fatty acid without using toxic solvents to retain the generally regarded as safe and effective (GRASE) nature of the product. To achieve this, we screened a number of GRASE compounds under varying conditions to arrive at the ammonium carboxylate salt of capric acid and L-arginine.
Capric acid is typically solid at room temperature (melting point 31.4 • C) with water solubility at 0.15 mg/mL at 20 • C with a pH 7.0 (as described previously) [39]. However, once compounded with L-arginine under specific conditions, the solubility of capric acid in water at 20 • C exceeds 150 mg/mL (1000-fold increase). Based on the known chemistry and synthesis conditions, upon compounding, the basic amine group on L-arginine deprotonates the carboxylic group in capric acid to form a highly stable, unreactive ammonium carboxylate salt ( Figure 1). This combination of capric acid and L-arginine in water is given the name GS-2.

Statistics
Statistical analyses (Student's unpaired two-tailed t-test) were performed with GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA).

GS-2 Development and Characterization
Capric acid is a 10-carbon saturated fatty acid with known antibacterial and antifungal properties [36][37][38]. However, esterifying fatty acids to solubilize them has been shown to reduce activity [24,30]. Our initial goal was to discover a method for solubilizing the fatty acid without compromising the native structure. We also needed to solubilize the fatty acid without using toxic solvents to retain the generally regarded as safe and effective (GRASE) nature of the product. To achieve this, we screened a number of GRASE compounds under varying conditions to arrive at the ammonium carboxylate salt of capric acid and L-arginine.
Capric acid is typically solid at room temperature (melting point 31.4 °C) with water solubility at 0.15 mg/mL at 20 °C with a pH 7.0 (as described previously) [39]. However, once compounded with L-arginine under specific conditions, the solubility of capric acid in water at 20 °C exceeds 150 mg/mL (1000-fold increase). Based on the known chemistry and synthesis conditions, upon compounding, the basic amine group on L-arginine deprotonates the carboxylic group in capric acid to form a highly stable, unreactive ammonium carboxylate salt ( Figure 1). This combination of capric acid and L-arginine in water is given the name GS-2.

L-arginine
Capric acid Ammonium carboxylate salt To further characterize the formulation and to confirm that no other products were produced upon compounding, GS-2 was separated into an aqueous fraction and a heptane fraction and subjected to 1 H and 13 C NMR. Spectra detected in the aqueous and heptane fractions were consistent with the number of protons and carbons present in L-arginine (Supplementary Figure S1) and capric acid (Supplementary Figure S2), respectively, as well as consistent with published work [40][41][42][43].
A GCMS analysis of the aqueous and heptane fractions of GS-2 was also performed to detect any low concentrations of secondary products. As observed with NMR, only L-arginine (or the thermal decomposition structure of arginine) was detected in the aqueous fraction, and only capric acid was detected in the heptane fraction (Supplementary Figure S3). These results suggest that there was no evidence of byproduct formation, To further characterize the formulation and to confirm that no other products were produced upon compounding, GS-2 was separated into an aqueous fraction and a heptane fraction and subjected to 1 H and 13 C NMR. Spectra detected in the aqueous and heptane fractions were consistent with the number of protons and carbons present in L-arginine (Supplementary Figure S1) and capric acid (Supplementary Figure S2), respectively, as well as consistent with published work [40][41][42][43].
A GCMS analysis of the aqueous and heptane fractions of GS-2 was also performed to detect any low concentrations of secondary products. As observed with NMR, only L-arginine (or the thermal decomposition structure of arginine) was detected in the aqueous fraction, and only capric acid was detected in the heptane fraction (Supplementary Figure S3). These results suggest that there was no evidence of byproduct formation, confirming that capric acid and arginine remained non-covalently bound in solution and that no new compounds were formed upon combining.
After compounding, the pH of the final solution of GS-2 at room temperature was 8.0-8.5, which appeared to be mediated by the basic buffering capacity of L-arginine. Acidification of GS-2 to pH 5 using 1 M HCl resulted in the disassembly of bonding between capric acid and L-arginine, with capric acid falling completely out of the solution. However, at pH 7, the solution of capric acid and L-arginine remained fully soluble with no evidence of turbidity. The final compounding of GS-2 was optimized with capric acid at 150 mg/mL with L-arginine in water. Subsequent testing showed that capric acid could be increased up to 300 mg/mL in the presence of L-arginine and maintain solubility. However, this increased the synthesis time from 2 h to over 48 h and, therefore, was not pursued.

Antibacterial and Antifungal Activity of GS-2
After compounding and characterizing its physical properties, the antimicrobial activity of GS-2 was tested against common bacterial pathogens: S. aureus, B. atrophaeus (surrogate for B. anthracis), C. butyricum (surrogate for Clostridium botulinum), C. difficile (vegetative and endospores), E. coli, K. pneumoniae, and P. aeruginosa, as well as the fungal pathogen C. auris. The Gram-positive pathogens S. aureus, B. atropheaus, C. butyricum, and C. difficile were most sensitive to GS-2, with MICs below 1 mg/mL and MBCs below 1.2 mg/mL ( Table 1). The Gram-negative rods (GNR) E. coli, K. pneumoniae, and P. aeruginosa displayed significantly higher MICs (3.75-7.5 mg/mL) and MBCs (3.75-10 mg/mL) ( Table 1), suggesting GS-2 was more potent against Gram-positive than Gram-negative pathogens. The MIC/MFC value observed against C. auris was similar to Gram-positives (Table 1), suggesting that GS-2 was also potent against fungal species. Testing with capric acid in DMSO against E. coli and K. pneumoniae did not produce an MIC or MBC, indicating that the combination of capric acid and L-arginine produced an enhanced antibacterial effect. The antibacterial activity of GS-2 was then tested against clinical isolates of four common bacterial pathogens: MRSA, S. pyogenes (Group A β-hemolytic strep), and CRE species K. pneumoniae and E. coli. The Gram-positive cocci (GPC) MRSA and S. pyogenes were most sensitive to GS-2, with S. pyogenes demonstrating the greatest sensitivity at an effective MBC of 0.78 mg/mL, whereas the Gram-negative rods (GNR) E. coli and K. pneumoniae displayed significantly higher MBCs (7.04 mg/mL and 8.04 mg/mL, respectively) ( Table 2). As a control, the use of L-arginine alone in water at 50 mg/mL did not produce an MIC or MBC in any of the bacteria tested. Capric acid in water at solubility (0.15 mg/mL) also failed to achieve an MIC or MBC in any of the four organisms tested here, again suggesting that the combination of capric acid and L-arginine conferred greater activity than the fatty acid alone. A criterion for disinfectants is a rapid time-kill. To determine the suitability of GS-2 as a disinfectant, we performed TGA disinfectant testing, standards relevant to Australia that test a disinfectant's activity against high concentrations of bacteria within 8 min. GS-2 passed this testing against E. coli but failed against MRSA ( Table 3), suggesting that GS-2 could rapidly kill Gram-negative bacteria but required a longer exposure time to kill Gram-positive bacteria. The activity of GS-2 was then tested against pathogens that cause significant disease and pose a high risk to be weaponized in circumstances such as bioterrorism. GS-2 was tested against B. thailandensis, a surrogate for B. pseudomallei that causes melioidosis, and B. anthracis, which causes anthrax. The treatment of B. thailandensis with GS-2 at 4.76 mg/mL resulted in a six-log decrease in growth and complete growth inhibition after 1 h and persisted 5 h after treatment ( Figure 2A). Interestingly, GS-2 activity was superior to the antibiotic meropenem, the current first-line therapy against melioidosis, highlighting that the difference in effect between GS-2 was the time-kill effect and the antibiotic effects of meropenem. However, after 24 h, growth slightly recovered, with an average of 200 CFU/mL detected in both GS-2-and meropenem-treated samples. Testing of GS-2 against B. anthracis vegetative Sterne cells showed a rapid (four-log) decrease in growth in the first 2 h ( Figure 2B). In contrast, treatment with ciprofloxacin, the first-line therapy for anthrax, resulted in a three-log reduction in B. anthracis vegetative cells.
K. pneumoniae 8.04 A criterion for disinfectants is a rapid time-kill. To determine the suitability of GS-2 as a disinfectant, we performed TGA disinfectant testing, standards relevant to Australia that test a disinfectant's activity against high concentrations of bacteria within 8 min. GS-2 passed this testing against E. coli but failed against MRSA ( Table 3), suggesting that GS-2 could rapidly kill Gram-negative bacteria but required a longer exposure time to kill Gram-positive bacteria. The activity of GS-2 was then tested against pathogens that cause significant disease and pose a high risk to be weaponized in circumstances such as bioterrorism. GS-2 was tested against B. thailandensis, a surrogate for B. pseudomallei that causes melioidosis, and B. anthracis, which causes anthrax. The treatment of B. thailandensis with GS-2 at 4.76 mg/mL resulted in a six-log decrease in growth and complete growth inhibition after 1 h and persisted 5 h after treatment (Figure 2A). Interestingly, GS-2 activity was superior to the antibiotic meropenem, the current first-line therapy against melioidosis, highlighting that the difference in effect between GS-2 was the time-kill effect and the antibiotic effects of meropenem. However, after 24 h, growth slightly recovered, with an average of 200 CFU/mL detected in both GS-2-and meropenem-treated samples. Testing of GS-2 against B. anthracis vegetative Sterne cells showed a rapid (four-log) decrease in growth in the first 2 h ( Figure 2B). In contrast, treatment with ciprofloxacin, the first-line therapy for anthrax, resulted in a three-log reduction in B. anthracis vegetative cells.

GS-2 Exhibits Antiviral Activity
For GS-2 to be considered an appropriate broad-spectrum antimicrobial, it must also have activity against viruses. To determine its antiviral activity, GS-2 was tested against coronavirus MHV-1, a SARS-CoV-2 surrogate. GS-2 achieved a 4.92-log reduction in viral titer after 10 min of exposure against MHV-1 (Table 4). This showed that GS-2 was effective against a coronavirus, supporting its use in surface decontamination against SARS-CoV-2. Table 4. Viral testing of 15 mg/mL GS-2 against COVID-19 surrogate using TMCV 006 ASTM E1053 methods for 10 min of exposure time; n = 3.

GS-2 Performs As an Effective Surface Sanitizer in Real-World Settings
Based on the antibacterial, antifungal, and antiviral activity of GS-2, the efficacy of GS-2 on surfaces was further tested. To test the effect of GS-2 in a real-world environment, two comparative commercial buildings were sanitized for two weeks with either GS-2 (15 mg/mL) or with a commercially available hospital-grade disinfectant containing 0.42% BAC according to label instructions. After two weeks (on day 15), five high-traffic touchpoints in each building were swabbed and cultured before sanitizing on day 15, 90 min after sanitizing, and 4 h after sanitizing ( Figure 3A). The first swab on day 15 before final sanitizing demonstrated that the building sanitized with 0.42% BAC for the previous 14 days had, on average, 5.6-fold more CFUs on touch-points than the building sanitized with GS-2, although the differences were not statistically significant (p = 0.18; Figure 3B). Ninety minutes after sanitizing on day 15, surfaces sanitized with 0.42% BAC hospital disinfectant had, on average, 20.3 times more CFUs than the GS-2-sanitized surfaces (p = 0.0087; Figure 3C). GS-2 activity appeared to persist longer than the BAC-containing disinfectant, and 4 h after sanitizing, there was a 7.9-fold reduction in CFUs recovered from GS-2-sanitized surfaces compared to BAC-treated surfaces (p = 0.17; Figure 3D). The authors acknowledge that these results are not wholly conclusive; there are variables in a 'real-world' study that could potentially bias these results, such as traffic through a building on a particular day and hand hygiene between people in buildings, and this is a single study between two buildings. However, the findings demonstrate that GS-2, under the tested conditions, provided antimicrobial activity at or better than a currently approved hospital-grade disinfectant.

GS-2 Poses No Toxic Effects and Is Rapidly Cleared In Vivo
Whilst capric acid is naturally found in foods such as coconut and palm kernel oil and L-arginine is a conditionally essential amino acid, we sought to confirm that there were no toxic effects of GS-2. Sprague-Dawley Rats were systemically challenged with GS-2 at 154.2 mg/mL at 10 mL of this solution per kg. The peak serum concentration of GS-2 (Tmax) was reached after 1.3 h, and capric acid was almost completely cleared from the rats after 24 h (Figure 4). We did not observe any abnormal behavior or any overt

GS-2 Poses No Toxic Effects and Is Rapidly Cleared In Vivo
Whilst capric acid is naturally found in foods such as coconut and palm kernel oil and L-arginine is a conditionally essential amino acid, we sought to confirm that there were no toxic effects of GS-2. Sprague-Dawley Rats were systemically challenged with GS-2 at 154.2 mg/mL at 10 mL of this solution per kg. The peak serum concentration of GS-2 (T max ) was reached after 1.3 h, and capric acid was almost completely cleared from the rats after 24 h (Figure 4). We did not observe any abnormal behavior or any overt toxicity, even at this systemic dose, which very likely exceeded any potential environmental exposure. Given the fact that capric acid and L-arginine are commonly found in the diets of mammals, the lack of toxicity associated with high systemic exposure was not wholly unexpected.

The Addition of Thymol Improves Antimicrobial Activity of GS-2
Data to this point demonstrate that GS-2 provided potent, broad-range antimicrobial activity. However, our findings clearly indicated that GS-2 required longer exposure times to show activity against Gram-positive bacteria, such as S. aureus (when compared to the Gram-negative rods), and thus, was not suitable as a disinfectant due to the longer exposure times required. To address this, we tested several naturally occurring GRASE compounds that have previously been reported as having significant antimicrobial activity. After extensive screening, we found that GS-2 formed a stable solution with thymol. Thymol is a naturally occurring 10-carbon monoterpene phenol derived from the plant Thymus vulgaris that exhibits antibacterial properties.
To test the antimicrobial activity of this GS-2 and thymol formulation, this formulation, as well as GS-2 alone and thymol alone, was tested with a short exposure time of 8 min to represent fast activity. Both 5.0 mg/mL thymol in DMSO and 30 mg/mL capric acid in DMSO were not able to prevent bacterial growth of MRSA or CRE with E. coli in this short exposure time (Table 5). However, the combination of thymol (5.0 mg/mL) and GS-2 (30 mg/mL) was successful in completely inhibiting the growth of both MRSA and E. coli within 8 min (Table 5).

The Addition of Thymol Improves Antimicrobial Activity of GS-2
Data to this point demonstrate that GS-2 provided potent, broad-range antimicrobial activity. However, our findings clearly indicated that GS-2 required longer exposure times to show activity against Gram-positive bacteria, such as S. aureus (when compared to the Gram-negative rods), and thus, was not suitable as a disinfectant due to the longer exposure times required. To address this, we tested several naturally occurring GRASE compounds that have previously been reported as having significant antimicrobial activity. After extensive screening, we found that GS-2 formed a stable solution with thymol. Thymol is a naturally occurring 10-carbon monoterpene phenol derived from the plant Thymus vulgaris that exhibits antibacterial properties.
To test the antimicrobial activity of this GS-2 and thymol formulation, this formulation, as well as GS-2 alone and thymol alone, was tested with a short exposure time of 8 min to represent fast activity. Both 5.0 mg/mL thymol in DMSO and 30 mg/mL capric acid in DMSO were not able to prevent bacterial growth of MRSA or CRE with E. coli in this short exposure time (Table 5). However, the combination of thymol (5.0 mg/mL) and GS-2 (30 mg/mL) was successful in completely inhibiting the growth of both MRSA and E. coli within 8 min ( Table 5).
Testing of GS-2 and thymol against S. aureus, E. coli, K. pneumoniae, and P. aeruginosa resulted in 2-4-fold lower MICs and MBCs (Table 6) when compared to GS-2 alone ( Table 1). The largest differences observed in the MICs and MBCs were against E. coli, K. pneumoniae, and P. aeruginosa, indicating that the addition of thymol improved activity against Gramnegative pathogens when exposed for 24 h.  To confirm the viricidal activity of this new formulation, GS-2 with thymol was tested against HSV-1, poliovirus, and MHV-1 with a 10 min exposure time using the TMCV 006 ASTM E1053 standard [34]. Testing of GS-2 with thymol against HSV-1, poliovirus, and MHV-1 produced > 6.5-log 10 , 3.0-log 10 , and 5.42-log 10 reductions of viral growth, respectively (Table 7). Interestingly, the GS-2 with thymol formulation demonstrated activity against the non-enveloped virus of poliovirus, a property commonly lacking from current surface antimicrobials and disinfectants [11,12]. Overall, GS-2 with thymol demonstrated broad antibacterial and antiviral activity within 10 min of exposure. Table 7. Testing of GS-2 (30 mg/mL) with thymol 5 (mg/mL) for surface viricidal activity according to the TMCV 006 ATSM E1053 method for 10 min of exposure; n = 3.

Virus Log 10 Reduction of Virus
HSV-1 >6.5 Poliovirus 3.0 MHV- 1 5.42 After further testing, we refined the ratio of GS-2 to thymol to optimize the antibacterial activity. Based on our testing, we arrived a ratio of 2:1 (GS-2:thymol) that provided optimal activity against both GNRs and Gram-positive cocci (GPC). As previously mentioned, GS-2 alone had substantial, rapid activity against GNRs; however, the formulation of ratios that moved away from the 2:1 ratio significantly extended the time required to kill GPCs. GS-2 at 15 mg/mL and thymol at 7.5 mg/mL successfully passed hospital-grade disinfectant testing using the EN13727 standard at 5 min of exposure under dirty conditions, achieving a >five-log reduction of growth for S. aureus, E. hirae, and P. aeruginosa (Table 8). Interestingly, a ratio of 6:1 (GS-2 to thymol) did not successfully pass this EN13727 testing, which suggested that the ratio of actives in the formulation is critical.

GS-2 with Thymol Remains Active on Surfaces
Based on the results of the building trial, it was hypothesized that GS-2 and the GS-2 with thymol formulation could bind to surfaces and retain activity over extended periods of time. To test this hypothesis, sterile slides were coated with the GS-2 and thymol formulation ( Figure 5A) and left uncovered on a benchtop for 0, 15, 30, and 60 days before inoculating with MRSA or P. aeruginosa. After 1 h of exposure to GS-2 with thymol, the viable bacteria were recovered, cultured, and counted. Surprisingly, the average MRSA recovered from slides treated with GS-2 and thymol after 60 days was 3.8 CFU/mL compared to the average of >460,000 CFU/mL recovered from the water control slides ( Figure 5B). Similarly, the average P. aeruginosa recovered from slides treated with GS-2 and thymol after 60 days was 8.8 CFU/mL compared to the >399,000 CFU/mL recovered from untreated controls ( Figure 5C). This was greater than a 99.99% reduction in recovered bacteria relative to the controls. These data suggest that GS-2 with thymol could persist on surfaces and maintain antibacterial activity for at least 60 days. inoculating with MRSA or P. aeruginosa. After 1 h of exposure to GS-2 with thymol, the viable bacteria were recovered, cultured, and counted. Surprisingly, the average MRSA recovered from slides treated with GS-2 and thymol after 60 days was 3.8 CFU/mL compared to the average of >460,000 CFU/mL recovered from the water control slides (Figure 5B). Similarly, the average P. aeruginosa recovered from slides treated with GS-2 and thymol after 60 days was 8.8 CFU/mL compared to the >399,000 CFU/mL recovered from untreated controls ( Figure 5C). This was greater than a 99.99% reduction in recovered bacteria relative to the controls. These data suggest that GS-2 with thymol could persist on surfaces and maintain antibacterial activity for at least 60 days.

Discussion
The present study described the scientific development of an antimicrobial, moving from initial characterization to standard antimicrobial testing for efficacy and completing with the development of a novel surface disinfectant. The antimicrobial activity of fatty

Discussion
The present study described the scientific development of an antimicrobial, moving from initial characterization to standard antimicrobial testing for efficacy and completing with the development of a novel surface disinfectant. The antimicrobial activity of fatty acids is well-known, with multiple studies examining their mechanisms of action and possible utility in controlling microbial contamination. However, a primary issue with fatty acids is very low solubility in water [44].
In this study, we sought to overcome these issues by combining capric acid with L-arginine to produce a highly water-soluble fatty acid formulation that was effective against a range of bacteria, fungi, and viruses. In real-world testing, GS-2 demonstrated surface sanitizing efficacy over a current hospital-grade disinfectant. With the addition of thymol to GS-2, a rapid, robust antimicrobial was created that was active against bacteria and viruses in vitro and retained antibacterial properties on surfaces for at least two months.
Low fatty acid solubility in water has previously limited the use of fatty acids as antimicrobials. By adding L-arginine, the solubility of capric acid could be increased to 300 mg/mL. We proposed that capric acid, when combined with L-arginine, formed an ammonium carboxylate salt and that this reaction was what allowed for the high solubility in water. We demonstrated that no secondary compounds were formed upon combining these two molecules. This was not surprising since, for an amide bond to form between arginine and capric acid, the components need to be heated above 100 • C to drive off water or require a coupling reagent [45], processes which were not performed during the compounding of GS-2 components. Furthermore, reducing the pH of GS-2 in the solution resulted in the separation of the components, further supporting the formation of a soluble salt.
The mechanism of action for antibacterial fatty acids has been defined in previous studies to be heavily dependent on three critical processes: (1) increased membrane permeability due to pore formation, leading to cell lysis; (2) attack of bacterial cell membranes via the electron transport chain, decreasing the production of ATP; and (3) inhibition of membrane enzymatic activities, such as glucosyltransferase, that inhibit nutrient uptake [46][47][48][49]. Interestingly, the results presented here showed that GS-2 was up to ten times more potent against GPCs (MRSA and S. pyogenes) than GNRs (E. coli and K. pneumoniae). However, the required exposure time to show activity against GPCs was much longer (24 h) compared to GNRs. This finding is unusual as it is in direct contradiction with Kabara et al. (1980), where fatty acids with more than eight carbons were found not to inhibit GNR growth [50]. It is possible that the effect against GNRs observed here was due to the chemical structure of the fatty acid in the solution and a lack of required excipients needed to solubilize the fatty acid that may have improved fatty acid activity in this study. Nonetheless, GS-2 still showed good potency against hardy environmental bacteria, as well as potential bioterrorism agent surrogates B. anthracis Sterne and B. thailandensis.
During developmental testing, increasing the concentration of GS-2 in the solution did not significantly improve the time-kill against GPCs, suggesting that the mechanism of action against GPCs was relatively independent of the concentration. This effect may be due to wall teichoic acids (WTAs) that attach to the peptidoglycan cell wall of S. aureus, which as a result, may require more time for fatty acids to act [51]. Although the cell walls of GNRs are considerably more complex than GPCs, WTAs are not typically found on GNR cell walls [51], which could explain why, at equivalent concentrations, GNRs were more susceptible than GPCs over shorter timespans.
The addition of thymol to GS-2 significantly improved the activity of GS-2 against GPCs. GS-2 with thymol demonstrated superior time-kill activity against S. aureus compared to GS-2 alone but did not have a remarkable improvement on the efficacy or time-kill effect for E. coli or K. pneumoniae. Thymol was previously shown to have bacteriostatic effects against S. aureus at 0.31 mg/mL (MIC) and E. coli at 5.0 mg/mL (MIC) when exposed for longer periods of time (18-24 h) [52]. However, in disinfectant testing procedures where exposures are relatively short, thymol at 5.0 mg/mL (in DMSO) was unable to achieve a bactericidal effect. However, when 5.0 mg/mL thymol was combined with GS-2, it was able to kill both S. aureus and E. coli in timeframes acceptable for use as a surface disinfectant. This suggests that thymol may act synergistically with GS-2 to deliver bactericidal activity after just 8 min of exposure; however, further studies are required to validate this effect and the mechanism that produces it.
Like the effect against GNRs, GS-2 with thymol only showed a modest improvement in efficacy compared with GS-2 against the SARS-CoV-2 surrogate MHV-1 (from 4.92-to 5.42-log reduction of viral titer after treatment). Doubling the concentration of GS-2 and adding thymol did not produce a clear synergistic effect against the virus but rather a modest additive effect. Whilst thymol has been described as having antiviral activity, high concentrations are required, or cells need to be pre-treated with thymol before infection to observe an effect [53,54]. Thus, it is not surprising that only a modest improvement in the antiviral activity of GS-2 was observed with the addition of thymol here. Nevertheless, GS-2 with thymol still demonstrated activity against three different viruses, including nonenveloped poliovirus, which is commonly resistant to other disinfectants. This indicated that GS-2 with thymol may be a suitable antiviral agent over a broad range of virus types.
The testing performed with GS-2 and, subsequently, GS-2 with thymol supported several possible advantages of this fatty acid formulation as a multipurpose antimicrobial sanitizer, particularly over quat-based products. Firstly, GS-2 displayed activity against C. auris, an emerging pathogen that is considered extremely difficult to eradicate with current disinfectant products [55]. In this study, GS-2 and GS-2 with thymol also demonstrated efficacy against C. difficile, P. aeruginosa, and poliovirus, all of which are considered problematic for quats [55][56][57]. It is also worth noting that the concentrations of GS-2 (capric acid; 15-30 mg/mL) and thymol (5-7.5 mg/mL) used in this study are comparable to the concentrations of BAC used in commercial disinfectants (1-30 mg/mL) [56], suggesting that GS-2 with thymol would be an acceptable alternative to quats.
Additionally, the results from the comparative commercial building study and the glass slide experiment suggested that GS-2 remained active on surfaces for up to 60 days. Considering the structure of the ammonium carboxylate salt formed, it was not surprising that GS-2 remained unreactive to degradation. Furthermore, given the fatty acid structure, GS-2 may be less sensitive to organic material found on high-touch-point surfaces than BAC; it has been thoroughly established that quats such as BAC lose efficacy on soiled surfaces [11,12], which may account for the higher number of viable bacteria recovered from surfaces sanitized with BAC seen here. A recent study testing GS-2 in a pork abattoir also found GS-2 to have superior antibacterial activity on surfaces compared to a quat-based disinfectant [58], further supporting its capability in a real-world setting. Together, these findings suggest that GS-2 may have broader bactericidal and virucidal activity against a range of pathogens when compared to currently available disinfectants.
Another advantage of GS-2 over other sanitizers is the safety profile; even high systemic doses of capric acid did not induce any toxicities in vivo. Like L-arginine and thymol, other fatty acids also have favorable safety profiles. Together, these results suggest utilizing amino acids to increase the water solubility of fatty acids may provide a platform for exploring, combining, and developing the antimicrobial activities of other fatty acids.
The current study presented scientific evidence regarding the development and testing of a novel antimicrobial ammonium carboxylate salt derived from a fatty acid and amino acid combination. Further evidence was presented that the addition of thymol, a 10-carbon monoterpenoid, to GS-2 enhanced the antimicrobial activity of GS-2, providing improved antimicrobial and disinfectant effects. Based on these data, GS-2 and GS-2 with thymol may have significant utility in the cleaning and disinfecting of surfaces, particularly in high-risk environments where drug-resistant pathogens pose a significant threat to human health. Finally, this platform of soluble fatty acids provides novel opportunities for the development of effective and safe broad-spectrum environmental antimicrobials.


s2fieldsofstudy: Biology Chemistry

text:
MiR-106a facilitates the sensorineural hearing loss induced by oxidative stress by targeting connexin-43

ABSTRACT Sensorineural hearing loss (SNHL) is a common clinical side effect resulted from the overusing of aminoglycoside antibacterial drugs, such as gentamicin. Oxidative stress is recently evidenced to be an important inducer for SNHL, which is reported to be associated with the knockdown of connexin-43. MiR-106a is recently found as a regulator of connexin-43. The present study aims to investigate whether miR-106a is a vital mediator in the development of SNHL. Firstly, upregulated miR-106a was observed in the peripheral blood sample of SNHL patients. Glucose oxidase (GO) was utilized to induce oxidative injury in isolated rat cochlear marginal cells (MCs), followed by introducing the miR-106a inhibitor. We found that the declined proliferation ability, increased apoptosis, and activated oxidative stress in GO-stimulated MCs were dramatically abolished by the miR-106a inhibitor, accompanied by the upregulation of connexin-43. The targeting correlation between miR-106a and connexin-43 was predicted and confirmed by the dual luciferase gene reporter assay. Furthermore, the regulatory effect of miR-106a inhibitor against the proliferation, apoptosis, and oxidative stress in GO-treated MCs were dramatically abolished by the knockdown of connexin-43. Gentamicin was utilized to establish the SNHL model in rats, followed by the treatments of antagomir-106a and antagomir-106a combined with carbenoxolone, an inhibitor of connexin-43. The alleviated pathological state, reduced apoptosis, and ameliorated oxidative stress in cochlea tissues were observed in antagomir-106a treated SNHL rats, which were dramatically reversed by the co-administration of carbenoxolone. Collectively, miR-106a facilitated the SNHL induced by oxidative stress via targeting connexin-43.

Introduction
Sensorineural hearing loss (SNHL) is one of the most common clinical diseases of ophthalmology and otorhinolaryngology, which is the abnormality of the auditory system to feel and process the sound resulted from various factors [1]. With the acceleration of population aging, hearing loss is the third chronic disease affecting the healthy life of the elderly [2] and the incidence of SNHL is still on the rise [3]. The main effect of adult hearing loss is communication obstruction, even psychological disorders. Currently, the treatment methods are limited and the application of assistive listening devices is an available solution. However, the therapeutic effect of auxiliary devices is associated with the number and role of residual cells in the cochlea [4]. Maintaining sufficient number of normal auditory cells is the prerequisite for keep normal auditory function. It is particularly important to prevent and treat the damage of cochlear cells. The pathogenesis of SNHL is complex and oxidative stress injury has been a hot topic in recent years [5]. Studies have shown that cochlea cells can be protected by antioxidants by preventing reactive oxygen species (ROS) from triggering caspase-mediated apoptosis and protecting mitochondrial membranes [6]. In SNHL animal models established by gentamicin, sharply increased level of ROS is observed in a short period of time, which can not be removed by timely antioxidant enzymes. Subsequently excessive free radicals are accumulated to produce malondialdehyde (MDA), which induces metabolic disorders on glucoses, proteins, and nucleic acids. The damages on cochlear tissues and cellular structures are finally induced to disrupt the normal functions [7,8]. Therefore, improving the activity of antioxidant enzymes, scavenging ROS, alleviating oxidative stress damage, and maintaining redox balance will be effective methods to mitigate the damage of cochlear cells, which is of great significance for the protection of hearing function [9]. MicroRNAs(miRNAs) are a group of RNA molecules with gene regulatory functions that have been discovered in recent years, the length of which is approximately 22 nucleotides [10,11]. Recently, several researches claimed the important role miRNAs play in the pathogenesis of SNHL. It is predicted by Li [12]that by screening differentially expressed miRNAs and mRNAs in samples from SNHL patients and health subjects, genes regulated by miR-34a, miR-548, miR-15a, miR-210, and miR-23a are found to paly critical roles in developing SNHL. Ebeid further claimed the function of miR-183 in the regeneration of sensory hair cells (HCs), the loss of which is an important inducer of SNHL, by targeting and regulating the expression level of Atoh1 [13]. However, rare sufficient experimental evidences for the specific function and regulatory mechanism of miRNAs in SNHL have been provided in recent years. It is reported that the downregulation of protein connexin-43 is closely associated with the pathogenesis of SNHL [14] and connexin-43 is considered as an important inhibitor of oxidative stress [15]. Recently, miR-106a is reported to be a targeting miRNA and regulator of connexin-43 [16].
Therefore, we suspected that miR-106a might be a critical mediator in the development of SNHL by impacting connexin-43. The present study will explore a potential pathological mechanism of SNHL and offer a biomarker for the diagnosis of SNHL by investigating the specific function of miR-106a in mediating glucose oxidase (GO) induced oxidative stress injury in rat cochlear marginal cells (MCs) and pathological symptoms in gentamicininduced SNHL rat model.

Collection of clinical SNHL samples
Peripheral blood samples were obtained from 10 pairs of SNHL patients resulted from overcommit of gentamicin and health subjects in Beijing University of Chinese Traditional Medicine Dongfang College, respectively. All SNHL patients and health subjects were aware of the purpose of sample collection and consented the project orally.

The isolation of rat cochlear MCs [17]
Following performed with inhaled anesthesia, 6 SD rats were administered with 75% ethanol, followed by removing the bilateral temporal bones and dissecting the stria vascularis from the apical to basal. Subsequently, the stria vascularis was cut into 0.5 mm slides, followed by being digested using the 0.1% type II collagenase. After 30 min incubation, cell suspension was centrifugated at 300 g for 5 min and cells were resuspended using the Epithelial Cell Medium-animal (EpiCM-animal, ScienCell, California, USA) under the condition of 37°C and 5% CO 2 . The isolated MCs were identified by determining the expression level of cytokeratin-18 (CK18) using the method of immunofluorescence [18].

Immunofluorescence assay [17]
MCs were collected and fixed utilizing the 4% paraformaldehyde on microslide for 10 min, which were further treated with triton-100 for 15 min. Following incubating with 5% BSA, cells were added with the mouse anti rat CK18 antibody (1:1000, biorbyt, Wuhan, China) at 37°C for 1.5 h, which were further added with the solution diluted with FITC Conjugated goat anti mouse antibody (1:500, Abcam, Cambridge, USA) at 37°C for 45 min. Lastly, the slides were sealed using nail polish after adding the DAPI solution. The fluorescence was observed under the fluorescence microscope (KEYENCE, Tokyo, Japan).

CCK-8 assay [18]
MCs were implanted in the 24-well plate, followed by treated with different strategies for 24 hours. Subsequently, each well was added with the CCK-8 solution to be incubated for 4 hours and the optical density (OD) at 450 nm was determined using the microplate reader (Bio-Chain, Shanghai, China).

Real time PCR [18]
TRIzol (Invitrogen, California, USA) was utilized to extract total RNAs from cells, followed by centrifugating at 16,000 g for 10 min. After being dissolved in ddH 2 O, the concentration of RNAs was quantified by determining the absorption at 260 nm, followed by transcribing RNAs to cDNAs with the reverse transcription kit (Applied Biosystems, Massachusetts, USA). Then, the reaction of RT-PCR was performed with the ABI 7500 Real-time PCR system (Applied Biosystems, Massachusetts, USA) utilizing the SYBR green (Invitrogen, California, USA). The procedure for the reaction was shown as below: 95°C for 1 min, 40 cycles of 95°C for 30 sec, 62°C for 60 sec, 72°C for 30 sec, and 72°C for 90 sec. The 2 −ΔΔCtn method was utilized to measure the expression level of mRNAs, with GAPDH for the normalization.The primers of human miR106a:XXXXXXXXXXXX; rat miR106a: XXXXXXX Analysis on apoptosis by using the flow cytometry [19] 3 × 10 6 cells were collected and the 5× Binding Buffer was diluted to 1× Binding Buffer with double steam water, followed by being used to resuspend cells. After adding 3 μL Annexin V-APC and 5 μL 7-AAD into each tube, cells were slightly mixed and incubated for 10 min. Then, 200 μL pre-cooled 1× Binding Buffer was added to each tube, followed by being loaded onto the ACEA NovoCyteTM (ACEA Biosciences, Hangzhou, China) for analysis.

DCFH-DA assay [20]
In brief, MCs were added with DCFH-DA reagent (ADANTI, Wuhan, China) to be incubated for 10 min and DMEM medium was used to wash the remaining reagent. The release of ROS was quantified with the ImageJ software after visualization utilizing the inverted fluorescence microscope (KEYENCE, Shanghai, China).

MDA, SOD and connexin43 measurement
The production of MDA and SOD in cells and tissues was determined using the commercial MDA and SOD assay kit (Spbio, Wuhan, China) referring to the manufacturer's instruction. The connexin43 in serum of human was detected by ELISA kit (Xinyu, Shanghai, China) referring to the manufacturer's instruction. [21,22] Proteins were obtained from cells and were then quantified with a BCA kit (Absin, Shanghai, China), followed by loaded onto a 12% SDS PAGE. After separating for 60 min, proteins were transferred onto the PVDF membrane, which was further incubated with 5% BSA. Then, the primary antibody against connexin-43 (1:800

Auditory brainstem-evoked response testing (ABR) [25]
The recording electrode was placed in the middle of the parietal skull, while the reference electrode and the ground electrode were placed under the skin of the donor ear and the contralateral ear, respectively. Stimulus parameters and test parameters were set as following: the stimulus sound was set as filtered click; the frequency of band-pass filter was 100-3000 Hz; the times of overlay was 512; the scanning duration was 10 ms; headset output; repetition rate was 10 times/second; the distance to the rat ear canal mouth was 0.5 cm. ABR was determined based on wave III.

H&E staining on cochlear tissues
Cochlear tissues were fixed with 4% paraformaldehyde and dehydrated using different concentrations of ethyl alcohol solution, followed by transparentized with xylene for 15 min. The paraffin was used to embed tissues for 60 min, followed by cutting into slides. After baking, dewaxing, and hydration, sections were dyed with hematoxylin solution for 3 min, which were further incubated with the hydrochloric acid for 15s and 3 washes. Samples were dyed with eosin solution for 3 min, which were washes, dehydration, transparentizing, and sealing. Lastly, images were taken under the inverted microscope (SUNNY OPTICAL, Zhejiang, China).

TUNEL assay
Slides were incubated with the working solution for 15 min at 37°C, followed by adding the 50 µL TUNEL reaction solutions (Solarbio, Beijing, China) to be incubated for 60 min. Then, samples were added with diaminobenzidine (DAB) for 30 minutes. Lastly, slides were visualized using the inverted microscope (SUNNY OPTICAL, Zhejiang, China).

Statistical analysis
Data achieved in the present study were presented as mean ± SD. The Student's t-test was utilized to analyze the difference between 2 groups and the one-way ANOVA method was utilized to analyze the differences among groups. P < 0.05 was taken as a significant difference.

Results
In this study, we explore the potential pathological mechanism of SNHL and offer a biomarker for the diagnosis of SNHL by investigating the specific function of miR-106a in mediating glucose oxidase (GO) induced oxidative stress injury in rat cochlear marginal cells (MCs) and pathological symptoms in gentamicin-induced SNHL rat model. Results showed that miR-106a might be a critical mediator in the development of SNHL by impacting connexin-43.

MiR-106a was significantly upregulated, but connexin43 was decreased in SNHL patients
We firstly measured levels of miR-106a and con-nexin43 in the blood sample collected from health subjects and SNHL patients. We found that a significantly higher levels of miR-106a (Figure 1(a)) were observed in SNHL patients (*p < 0.05), however, the protein of connexin43 (Figure 1(b)) was decreased compared with health subjects(p < 0.01).

The cytotoxicity of GO on isolated MCs
Firstly, the isolated MCs were identified by the positive expression of CK18 using the immunofluorescence assay (Figure 2(a)). The cell viability was further detected after MCs were added with GO (0, 1, 2.5, 5, 10, 20, 40, 80, and 160 mU/mL) to determine the optimized incubation concentration. When the concentration of GO was larger than 20 mU/mL, significantly declined cell viability of MCs was observed (Figure 2(b), **p < 0.01, ***p < 0.001). In the subsequent experiments, 20 mU/mL GO were introduced for the investigations as 20 mU/mL GO was the lowest concentration that induced the declined proliferation in MCs.

MiR-106a inhibitor repressed the apoptosis in GO-treated MCs
To investigate the impact of miR-106a on GOinduced apoptosis in MCs, MCs were treated with 20 mU/mL GO with or without inhibitor NC and miR-106a inhibitor, respectively. The OD values (Figure 3(a)) was declined in MCs by the stimulation of GO, which was greatly elevated by miR-106a inhibitor. The apoptotic rate of MCs (Figure 3(b)) was promoted from 7.1% to 39.3% by GO, which was slight changed to 40.2% by inhibitor NC and declined to 17.9% by miR-106a inhibitor. Additionally, Bax and caspase-3 (Figure 3(c)) were dramatically upregulated and Bcl-2 was greatly downregulated in the GO group and GO+ inhibitor NC group, which were significantly reversed by miR-106a inhibitor (**p < 0.01 vs. control, ##p < 0.01 vs. GO+ inhibitor NC). These data collectively implied that apoptosis in GO-treated MCs was alleviated by the miR-106a inhibitor.

MiR-106a inhibitor mitigated oxidative stress in GO-treated MCs
We further explored the influence of miR-106a on oxidative stress in MCs induced by GO. Significantly higher ROS level (Figure 3(d)) was observed in the GO group and GO+ inhibitor NC group, which was greatly repressed by the introduction of miR-106a inhibitor. Additionally, the concentration of MDA was dramatically elevated and that of SOD was greatly declined by the stimulation of GO with or without inhibitor NC, which were greatly reversed by miR-106a inhibitor (Figure 3(e-f)). Subsequently, the activity of the key pathway regulating oxidative stress was evaluated. Nrf2 and HO-1 (Figure 3(g)) were dramatically downregulated and Keap1 was greatly upregulated in the GO group and GO+ inhibitor NC group, which were abolished by the miR-106a inhibitor (**p < 0.01 vs. control, ##p < 0.01 vs. GO+ inhibitor NC). These results implied that GO-stimulated oxidative stress in MCs was mitigated by the miR-106a inhibitor.

MiR-106a regulated the expression of connexin-43 by targeting the 3ʹUTR region
Firstly, we checked the state of miR-106a/connexin-43 in above grouping. MiR-106a (Figure 4 (a)) was found significantly upregulated and connexin-43 (Figure 4(b)) was dramatically downregulated by the treatment of GO with or without inhibitor NC, which were greatly reversed by miR-106a inhibitor (**p < 0.01 vs. control, ##p < 0.01 vs. GO+ inhibitor NC). We suspected that a correlation between miR-106a and connexin-43 might be available, which was further identified using the dual-luciferase gene reporter assay. The predicted binding sequence between miR-106a and connexin-43 were shown in Figure 4(c). We found that in cells transfected with connexin-43-WT, no obvious difference was seen between the miR-NC and miR-106a mimic group. However, dramatically declined luciferase activity was observed in the miR-106a mimic group (**p < 0.01 vs. miR- Figure 2. The isolated rat cochlear MCs were identified and the incubation concentration of GO was determined. A. The isolated rat cochlear MCs were identified by checking the expression of CK18 using the immunofluorescence assay. B. The OD value was measured using the CCK-8 assay (**p < 0.01, ***p < 0.001). Scale bar: 50 μm. Figure 3. The apoptosis and oxidative stress in GO-treated MCs were ameliorated by the knockdown of miR-106a. MCs were treated with 20 mU/mL GO with or without inhibitor NC and miR-106a inhibitor, respectively. A. The OD value was checked utilizing the CCK-8 assay. B. The apoptotic rate was investigated by the flow cytometry assay. C. The level of Bcl-2, Bax, and caspase-3 was evaluated by the Western blotting assay. D. The DCFH-DA assay was utilized to determine the ROS level in MCs. E. The production of MDA was investigated utilizing the commercial kit. F. The production of SOD was investigated utilizing the commercial kit. G. The level of Nrf2, Keap1, and HO-1 was confirmed by the Western blotting assay (**p < 0.01 vs. control, ##p < 0.01 vs. GO+ inhibitor NC). NC). Considering above observation, we concluded that miR-106a targeted connexin-43 mRNA to regulate the expression of connexin-43.

Knockdown of connexin-43 canceled the inhibitory effects of miR-106a inhibitor against GO-induced apoptosis in MCs
To verify that miR-106a exerted the facilitating function in GO-induced injury in MCs, MCs were treated with 20 mU/mL GO and miR-106a inhibitor with or without a siRNA targeting connexin-43 (siR-connexin-43). Firstly, the declined OD value ( Figure 5 (a)) in the miR-106a inhibitor group was dramatically promoted by the co-introduction of siR-connexin-43. The apoptotic rate was slightly changed from 40.0% to 40.1% in GO-stimulated MCs by the treatment of inhibitor NC combined with siR-NC, while the apoptotic rate was greatly reduced to 16.5% in the GO+ miR-106a inhibitor group, which was reversed to 28.2% in the GO+ miR-106a inhibitor+ siR-connexin-43 group (Figure 5(b)). Additionally, in the miR-106a inhibitor group, significantly declined expression level of Bax and caspase-3, as well as the increased level of Bcl-2 ( Figure 5(c)), were observed, which were greatly reversed by siR-connexin-43 (**p < 0.01 vs. GO+ inhibitor NC+ siR-NC, ##p < 0.01 vs. GO+ miR-106a inhibitor). These data implied that miR-106a regulated the GO-induced apoptosis in MCs by mediating the expression of connexin-43.

Knockdown of connexin-43 abolished the inhibitory effects of miR-106a inhibitor against GO-induced oxidative stress in MCs
In additional to apoptosis, we suspected that the regulatory effect of miR-106a on oxidative stress was also associated with its inhibitory effect on the expression of connexin-43. We found that the significantly reduced ROS level ( Figure 5(d)) observed in the GO+ miR-106a inhibitor group was dramatically promoted by the co-treatment of siR-connexin-43. Additionally, the reduced production of MDA ( Figure 5(e)) and elevated concentration of SOD ( Figure 5(f)) in the GO+ miR-106a inhibitor group were dramatically reversed by siR-connexin-43. Lastly, upregulated Nrf2, connexin-43, and HO-1 and downregulated Keap1 ( Figure 5(g)) in the GO+ miR-106a inhibitor group were greatly abolished by siR-connexin-43 (**p < 0.01 vs. GO+ inhibitor NC+ siR-NC,  A. The OD value was checked utilizing the CCK-8 assay. B. The apoptotic rate was investigated by the flow cytometry assay. C. The level of Bcl-2, Bax, and caspase-3 was evaluated by the Western blotting assay. D. The DCFH-DA assay was utilized to determine the ROS level in MCs. E. The production of MDA was investigated utilizing the commercial kit. F. The production of SOD was investigated utilizing the commercial kit. G. The level of Nrf2, Keap1, and HO-1 was confirmed by the Western blotting assay (**p < 0.01 vs. GO+ inhibitor NC+ siR-NC, #p < 0.05 vs. GO+ miR-106a inhibitor, ##p < 0.01 vs. GO+ miR-106a inhibitor). #p < 0.05 vs. GO+ miR-106a inhibitor, ##p < 0.01 vs. GO+ miR-106a inhibitor). These results implied that miR-106a regulated the GO-induced oxidative stress in MCs by mediating the expression of connexin-43.

Pathological state in SNHL rats were alleviated by antagomir-106a
To confirm the function of miR-106a in SNHL, SNHL rats were administered with antagomir-NC, antagomir-106a, or antagomir-106a combined with carbenoxolone, respectively. Firstly, the increased ABR value (Figure 6(a)) in the Model+ antagomir-NC group was significantly reduced in the antagomir-106a group, which was greatly reversed by the co-treatment of carbenoxolone. Next step, the level of miRNA106a in SNHL rats was detected, the level of miR106a (Figure 6(b)) was increased in the Model+ antagomir-NC group, which was significantly reduced in the antagomir-106a group (p < 0.01). In the control group, the spiral ganglion cells in cochlea tissues were complete in shape and neatly arranged. The cell membrane was round or oval, the nucleoli was clear, and the chromatin was uniform. In the antagomir-NC group, the number of spiral ganglion cells in cochlea tissues was significantly reduced with different sizes, irregular and disordered arrangements, almost disappeared nucleus, and vacuolated degeneration. These pathological changes were significantly ameliorated in the antagomir-106a group. However, by the coadministration of carbenoxolone, the disordered arrangements, disappeared nucleus, and vacuolated degeneration were anew observed in spiral ganglion cells (Figure 6(c)). Additionally, increased TUNEL positive staining was observed in the antagomir-NC group, which was reduced by the treatment of antagomir-106a. However, compared to the antagomir-106a group, the TUNEL positive staining was anew enhanced in the antagomir-106a+ carbenoxolone group (Figure 6(d)). We further found that the upregulated Bax and caspase-3 ( Fig 8E) and the downregulated Bcl-2 in the antagomir-NC group were greatly reversed by antagomir-106a. However, compared to the antagomir-106a group, the expression level of Bax and caspase-3 was largely promoted and the expression level of Bcl-2 was greatly reduced in the antagomir-106a+ carbenoxolone group (**p < 0.01 vs. control, ##p < 0.01 vs. antagomir-NC, && p < 0.01 vs. antagomir-106a). These data suggested that the pathological state in SNHL rats were alleviated by antagomir-106a through regulating connexin-43.

Oxidative stress in SNHL rats were mitigated by antagomir-106a
We lastly evaluated the state of oxidative stress in cochlear tissues. We found that the elevated release of MDA (Figure 6(f)) and declined concentration of SOD (Figure 6(g)) in the antagomir-NC group were dramatically reversed by antagomir-106a. However, compared to the antagomir-106a group, significantly increased MDA level and reduced SOD level were observed in the antagomir-106a+ carbenoxolone group. Additionally, as the Figure 6(h) showed, the downregulated Nrf2 and HO-1, as well as the upregulated Keap1, were observed in the antagomir-NC group, which were greatly reversed by antagomir-106a. Compared to the antagomir-106a group, dramatically declined expression level of Nrf2 and HO-1 and promoted level of Keap1 were observed in the antagomir-106a+ carbenoxolone group (**p < 0.01 vs. control, ##p < 0.01 vs. antagomir-NC, & p < 0.05 vs. antagomir-106a, && p < 0.01 vs. antagomir-106a). These data suggested that the oxidative stress in SNHL rats were mitigated by antagomir-106a through regulating connexin-43.

Discussion
Cochlea is an organ that maintains high aerobic metabolism and produces a large amount of ROS while providing ATP for energy supply, which is thereby vulnerable to ROS oxidation [26]. Stria vascularis is an important structure located in the lateral wall of the cochlea, which a special stratified epithelium containing capillaries composed of three types of cells: MCs in the outer layer, intermediate cells in the middle layer, and basal cells in the inner layer. MCs are adjacent to the endolymph cavity, with Na + -K + -ATPase and Na + -K + channels located on the cell membranes, which are of great significance for maintaining the endolymph ionic environment and the cochlear micro-potential [27]. Once MCs are damaged by ROS, the energy supply of the inner ear will be disrupted [28]. The present study used MCs isolated from the rat cochlea tissue to establish the oxidative stress model under the stimulation of Figure 6. Pathological state and the level of oxidative stress in SNHL rats were alleviated by antagomir-106a. SNHL rats were administered with antagomir-NC, antagomir-106a, or antagomir-106a combined with carbenoxolone, respectively. A. The ABR values were recorded. B. The level of miRNA106a in SNHL rats was detected by RT-PCR. C. The pathological changes in cochlea tissues were determined by HE staining. D. The apoptosis in cochlea tissues was evaluated by the TUNEL assay. E. The level of Bcl-2, Bax, and caspase-3 was evaluated by the Western blotting assay. F. The concentration of MDA was measured using the commercial kit. G. The concentration of SOD was measured using the commercial kit.H. The expression level of Nrf2, Keap1, and HO-1 was evaluated by the Western blotting assay (**p < 0.01 vs. control, ##p < 0.01 vs. antagomir-NC, & p < 0.05 vs. antagomir-106a, && p < 0.01 vs. antagomir-106a). Scale bar: 100 μm.
GO, a reagent utilized to mimic the oxidative state [27].
Under a normal physiological state, the balance between the production and elimination of ROS was maintained in endothelial cells. However, when the ability of eliminating ROS declines, oxidative stress will be activated [29]. There were many research showed that Oxidative stress plays a role in the pathogenesis of noise-related hearing loss, as well as in drug-and aging-related hearing loss [9,30,31].MDA is the terminal output of lipid peroxidation induced by the action of oxygen free radicals on polyunsaturated fatty acids on biofilms. The level of MDA reflects the severity of free radical attack and can be used as an indicator to evaluate degree of oxidative stress [32][33][34]. Superoxide Dismutase (SOD) is an enzyme that catalyzes the superoxide anion radical disproportionation to generate oxygen and hydrogen peroxide. Declined activity of SOD is considered as a critical biomarker of oxidative stress [35]. We found that under the treatment of GO, classic symptoms of oxidative stress were observed in MCs, such as increased release of ROS, elevated MDA level, and declined SOD concentration, which was also reported by Kumar in H9c2 cells [36]. Additionally, in gentamicin-induced SNHL animal model, significant oxidative stress was observed in cochlea tissues. Similar results have been reported by Jiang [37] in 2016. These data further confirmed the important role of oxidative stress in SNHL.
MicroRNAs are small, non-coding, singlestranded RNAs, which have approximately 21-23 nucleotides [38]. There is increasing evidence that microRNA could regulated of gene expression in the post-developmental inner ear and contributed to the development of acquired hearing loss [38][39][40]. Resent study also showed that circulating microRNAs maybe new diagnostic biomarkers of hearing loss [41].MiR-106a is a miRNA that has been evidenced to be an important regulator in multiple diseases, such as diabetic nephropathy [42], gastric cancer [43], and gestational hypertension [44]. However, the function of miR-106a in SNHL is not clear. Recently, several reports claimed the regulatory effects of miR-106a against oxidative stress. Hu reported [45] that the ox-LDL-induced oxidative stress in endothelial cells could be reversed by the knockdown of miR-106a, indicating a facilitating effect of miR-106a against oxidative stress. However, an inhibitory effect of miR-106a against oxidative stress was reported by Wu [46]. Therefore, the role of miR-106a in regulating oxidative stress is controversial. The present study revealed that knockdown of miR-106a not only repressed oxidative stress in GOtreated MCs but also reversed oxidative stress in cochlear tissues in SNHL rats, accompanied by the alleviation of pathological state of SNHL rats, suggesting that in the development of SNHL, miR-106a functioned as an inhibitory mediator against oxidative stress.
Gap junction (GJ) is a channel between the membranes of adjacent cells, which allows the passage of substances with molecular weight less than 1000 Da, including some ions, second messengers, and nutrient metabolites [47]. GJ are mainly formed by the interlacing of two junctions, which are half-channels consisting of connexins, on adjacent cell membranes. Connexin 43, a member of the connexins family, belongs to type II connexin, which is the first connexin that is confirmed associated with deafness [14]. Zhang reported that the mild hearing loss could be induced by the downregulation of connexin 43 [48], which was supported by Wang [49] using a postnatal mice model. We found that the declined expression level of connexin 43 was both observed in GO-treated MCs and cochlear tissues of SNHL rats, suggesting connexin 43 might be protective to the development of SNHL. The correction between miR-106a and connexin 43 was further identified, which was consistent with Wang's report [16]. Verification experiments implied that the inhibitory effects of miR-106a inhibitor against oxidative stress and alleviating effects of antagomir-106a against both GO-induced injury in MCs and SNHL symptoms were dramatically abolished by the knockdown of connexin 43. Actually, the inhibitory effects of connexin 43 against oxidative stress have already been reported in several researches [15,50], which together with our finding confirmed the important role of miR-106a/connexin 43 in oxidative stress injury in MCs and SNHL. In future work, the upstream regulatory mechanism of miR-106a will be further investigated to explore more information on the pathogenesis of SNHL.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by 2022 National Double first-class discipline (otolaryngology combined TCM with western medicine) promotion project of Beijing University of Chinese Medicine.